Plasmodium falciparum is the most virulent form of malaria, causing ~ 0.5 million deaths per year. Success with patient treatment and a malaria eradication campaign depends significantly on our ability 1) to detect new infections as early as possible in the field/rural areas, including asymptomatic cases, preferably using a small amount of blood (e.g. from a finger-prick) and 2) to clearly discriminate between current/new infection and past infections. Our ultimate goal is to build an inexpensive device that will be fully operational in the field with minimal training and capable of highly sensitive and selective identification of new malaria infections. We propose a three-phase study, with the Phase I (current proposal) focused on identification of the appropriate molecular targets and the development of a sensitive method of target detection that would discriminate between current and past infections. This project will be performed by Cascade Biosystems, Inc., in collaboration with scientists from Florida Atlantic University, Muhimbili University of Health and Allied Sciences, Tanzania, and Uppsala University, Sweden. The detection approach is a recently developed technology that is based on a cascade of enzymatic reactions catalyzed by restriction endonucleases (RCEA, restriction cascade exponential amplification). RCEA can detect attomolar concentrations of target DNA, comparable in terms of sensitivity to quantitative PCR; more importantly it is isothermal and does not require complex instrumentation or expensive reagents. In the Phase I we will adapt RCEA for the diagnostics of malaria. Specific Aims of the Phase I of the proposal are designed to demonstrate a proof-of-principle: the ability of RCEA approach to detect malaria at high sensitivities comparable to qPCR, and to discriminate between current and past infection. Aim 1. Identify appropriate molecular targets for detection of P. falciparum infection in culture using the RCEA approach and compare sensitivity of the RCEA detection to the standard qPCR approach. Parasites growing in culture will be used to simulate and test a wide range of parasite densities. Milestone: The RCEA assay developed specifically for detection of malaria targets in 100 ?l of human blood, comparable to qPCR in terms of sensitivity. Aim 2. Determine the potency of the developed RCEA assay for various gene targets to detect current and past infections in field samples. Field samples with various parasitemia will be tested for diagnosis of malaria using methods established in Aim 1. Various time points between current and past infection will be simulated using mixtures of samples from untreated and drug-treated patients. Milestone: the standard operation procedure for detection of current malaria infection by the RCEA. Successful completion of these aims will provide a proof-of-principle and a strong basis for the Phase II: (i) development of a fully integrated device based on electrochemical signal detection for field application of the developed RCEA assay, and (ii) the device testing in the field in malaria endemic areas. This, in turn, will be the basis for the Phase III focusing on miniaturization and commercialization of the device.